(A) Top: Representative current traces for the HHHEH chimera and its single point mutants. Black current traces are elicted by pulses to depolarizing potentials whereas are red current traces are elicted by pulses to hyperpolarizing potentials. Test pulses range from −150 mV to 50 mV from a holding potential of −40 mV (Histidine, Glutamine, Alanine, Glycine), −50 mV (Serine), or −100 mV (Isoleucine, Valine, Proline). Scale bars represent 5 µA (vertical) and 200 ms (horizontal). Color coding and scale bars are the same throughout the figure. Bottom: Relative PO vs voltage plots for the S272 mutants in the HHHEH background. The black trace in each plot represents the original chimera with a serine at 272. Error bars represent Standard Deviation from n = 3 (Isoleucine and Histidine), 4 (Proline, Alanine and Glutamine) and 6 (Valine) from independent measurements. (B) Gating polarity index (PO-150 mV/PO50 mV) were plotted against the transmembrane helical propensity (left), Kyle-Doolittle (KD) hydrophobicity index (middle), and turn propensity (right) of amino acids according to those described previously (see Figure 5). (C) Representative current traces for the HHHEΔC SD chimera and its single point mutants. Test pulses range from −150 mV to 50 mV from a holding potential of −40 mV (Glycine) or −90 mV (all others). Bottom: Relative PO vs voltage plots for the S272 mutants in the HHHEΔC SD background. Error bars represent Standard Deviation from n = 5 (Histidine, Asparagine and Glycine), 6 (Leucine and Phenylalanine), or 4 (Proline) independent measurements. (D) Left: Structural overlay of the hHCN1 and rEAG1 voltage sensors with the position of G362 highlighted as spheres. Middle: Representative current trace for hEAG1 G362S measured in two-electrode voltage clamp. Due to the high conductance of this mutant at negative membrane potentials, current recordings in high external potassium resulted in clamp errors, thus ND96 solution was used externally (2 mM potassium). Right: Open probability vs. voltage relationships for hEAG1 and the G362S mutant. Error bars represent standard deviation.